Removing the mustard oil bomb from seeds: transgenic ablation of myrosin cells in oilseed rape (Brassica napus) produces MINELESS seeds

Abstract

Many plant phytochemicals constitute binary enzyme–glucoside systems and function in plant defence. In brassicas, the enzyme myrosinase is confined to specific myrosin cells that separate the enzyme from its substrate; the glucosinolates. The myrosinase-catalysed release of toxic and bioactive compounds such as isothiocyanates, upon activation or tissue damage, has been termed ‘the mustard oil bomb’ and characterized as a ‘toxic mine’ in plant defence. The removal of myrosin cells and the enzyme that triggers the release of phytochemicals have been investigated by genetically modifying Brassica napus plants to remove myrosinase-storing idioblasts. A construct with the seed myrosin cell-specific Myr1.Bn1 promoter was used to express a ribonuclease, barnase. Transgenic plants ectopically expressing barnase were embryo lethal. Co-expressing barnase under the control of the Myr1.Bn1 promoter with the barnase inhibitor, barstar, under the control of the cauliflower mosaic virus 35S promoter enabled a selective and controlled death of myrosin cells without affecting plant viability. Ablation of myrosin cells was confirmed with light and electron microscopy, with immunohistological analysis and immunogold-electron microscopy analysis showing empty holes where myrosin cells normally are localized. Further evidence for a successful myrosin cell ablation comes from immunoblots showing absence of myrosinase and negligible myrosinase activity, and autolysis experiments showing negligible production of glucosinolate hydrolysis products. The plants where the myrosin defence cells have been ablated and named ‘MINELESS plants’. The epithiospecifier protein profile and glucosinolate levels were changed in MINELESS plants, pointing to localization of myrosinases and a 35 kDa epithiospecifier protein in myrosin cells and a reduced turnover of glucosinolates in MINELESS plants.

Engineering Glucosinolates in Plants: Current Knowledge and Potential Uses

“…Hence, regulated expression of GSL biosynthesis and metabolism will provide an insight for controlling insect, herbivore damage, and disease prevention. Recently, Borgen et al [224] developed transgenic B. napus plants with the seeds lacking the myrosin cells (mine less plants), providing a good model to study plant defense systems and other related functions. More efforts in this direction could lead us to new avenues in plant-pathogen interactions and stress tolerance studies.…”

Semiochemical-based alternatives to synthetic toxicant insecticides for pollen beetle management

“…The glucosinolate defence system in OSR with their breakdown products and volatile profiles offers great prospects for manipulation to achieve increased pest control . Transgenic OSR has been produced with low myrosinase activity (Borgen et al 2010) which consequently reduces the production of glucosinolate hydrolysis products (mainly isothiocyanates). Compared to wild-type, these low isothiocyanate releasing seedlings were more preferred in free-choice experiments by generalist aphid species but less preferred by specialist aphid species (Borgen et al 2012).…”

Leaf and root glucosinolate profiles of Chinese cabbage (Brassica rapa ssp. pekinensis) as a systemic response to methyl jasmonate and salicylic acid elicitation

“…Glucosinolates (GSs) are one of the major defensive chemicals used by cruciferous plants to mediate plant innate immunity. Upon tissue damage occurring, GSs are hydrolyzed into bioactive isothiocyanates, thiocyanates, or nitriles by the action of myrosinase (Ratzka et al, 2002;Borgen et al, 2010). These GS hydrolysis products are toxic to fungi, bacterial pathogens, or herbivores (Agrawal and Kurashige, 2003;Wittstock et al, 2003;Brader et al, 2006;Burow et al, 2006;Mumm et al, 2008;Redovniković et al, 2008).…”